The benefits of phase imaging with the AFM

Atomic force microscopes are unique in that they can generate nanomechanical information through the direct interaction of the tip with the sample. The most straightforward, qualitative access to this information is by imaging in vibrating mode and simultaneously collecting topography and phase data. This interaction between the surface and the vibrating tip causes a phase shift in the oscillation, which correlates with surface hardness/stiffness. While only qualitative, this easy access to mechanical information can rapidly give insights into the distribution of composites in biomaterials.

polymer substrate before cleaning topo sidepolymer substrate before cleaning phase side
The AFMWorkshop HR AFM is the most affordable and robust AFM to give users access to subnanometer imaging capabilities.

Topography vs. phase at the nanoscale. The AFMWorkshop HR-AFM is a sample-scanning atomic force microscope with a top view microscope and side-view optics that can accommodate a liquid cell for sub-nanometer imaging projects (<35 pm noise).

Surface modification of biomaterials

Many life science applications use modified and chemically functionalized surfaces. The AFM excels in quality and process control to verify the raw materials and surface coverage of each synthetic step. In this example we were comparing the flatness and cleanliness of commercially available polymer substrates. Phase imaging proved to be extremely useful in identifying the types of contaminants and evaluating possible approaches for their removal.

AFM topography image of a polymer substrate before cleaning, showing particulate surface contamination.
AFM phase image of a polymer substrate before cleaning, revealing a difference in stiffness of a droplet contamination

Left: topography image of polymer substrate before cleaning. Right: corresponding phase image, identifying two distinct contaminants on the surface.

The topography image suggests that there is a flat surface in the background that is covered in contaminants. The phase image indicates that these consist of polymer fragments (identical phase) and softer droplets (phase shift).

AFM topography image of a polymer substrate after rinsing with ethanol, leaving only small particulates on the surface.
AFM phase image of a polymer substrate after rinsing with ethanol, leaving only polymer particulates with identical stiffness behind.

Left: topography image after ethanol rinse. Right: corresponding phase image, showing removal of the droplet contaminant.

Rinsing the substrate with ethanol removes the droplet contaminant but is not sufficient to remove the polymer particulates.

AFM topography image of a polymer substrate after wiping with ethanol, which removed most particulates but scratched the surface.
AFM phase image of a polymer substrate after wiping with ethanol, leaving only polymer debris with identical stiffness behind.

Left: topography image after ethanol wipe showing scratching of the surface but removal of contaminants. Right: corresponding phase image.

Wiping the substrate with ethanol removes both contaminants but scratches the surface. Nevertheless, this means the polymer contaminants are fragments that are not covalently bound to the surface and can be removed.

polymer before cleaning statistical quantities
polymer rinsed statistical quantities
polymer wiped statistical quantities

Extracted statistical quantities from topography scans before cleaning, after ethanol rinse and after ethanol wipe.

Since AFM scans are quantitative 3D datasets we can extract statistical quantities of these surfaces with one click to easily compare procedures. The quantitative aspect of AFM datasets can also be used to create and extract precise maps of particulates for additional analysis.

polymer substrate before cleaning cropped area
polymer substrate before cleaning cropped area masked
polymer substrate before cleaning cropped area extracted mask

Using the quantitative topography data to create and extract masks for additional measurements or image-based analysis.

Hydrogel characterization

Hydrogels are another important class of biomaterial composites with a wide range of applications. Because of their high water content and the structural changes they undergo when water is added or removed, hydrogels are very difficult to characterize in the vacuum of an electron microscope. In addition, their mechanical properties often are key to their function and again vary with their water content, making AFM phase imaging uniquely suited for their characterization.

The samples below are part of a project on a two-component hydrogel made by SPARK IP.

AFM topography image of a two component hydrogel showing showing a complex topography with pores and small surface features
AFM phase image of a two-component hydrogel showing mechanical differences of the two components

Left: topography image of a two-component hydrogel. Right: corresponding phase image. Hydrogel made by SPARK IP.

Even with just two main components, the topography image of this hydrogel sample shows many surface features in “flat” areas, such as pores and nanoparticles. The phase image beautifully illustrates the complex intermixing of the components, creating domains of various shapes and sizes.

Literature on AFM phase imaging in biomaterials research

  • Nagao, E., & Dvorak, J. A. (1999). Phase imaging by atomic force microscopy: analysis of living homoiothermic vertebrate cells. Biophysical journal76(6), 3289–3297. https://doi.org/10.1016/S0006-3495(99)77481-3

  • Whitehead, A. J., Kirkland, N. J., & Engler, A. J. (2021). Atomic Force Microscopy for Live-Cell and Hydrogel Measurement. Methods in molecular biology (Clifton, N.J.)2299, 217–226. https://doi.org/10.1007/978-1-0716-1382-5_16

  • Joshi, J., Homburg, S. V., & Ehrmann, A. (2022). Atomic Force Microscopy (AFM) on Biopolymers and Hydrogels for Biotechnological Applications-Possibilities and Limits. Polymers14(6), 1267. https://doi.org/10.3390/polym14061267